Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR CULTURING ANTIBODY SECRETING CELLS
RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. 119(e) to
U.S.
Provisional Application No. 63/199,560, filed January 8, 2021, the entire
contents of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
This invention relates to methods for culturing antibody secreting cells for
antibody
production using a hollow fiber bioreactor, and related compositions and
methods.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that antibody
secreting cells
can be successfully cultured on a cell monolayer using a hollow fiber
bioreactor system. Cells
cultured in accordance with the methods described here display a surprising
longevity
compared to other ex vivo cultures systems. Thus, in one aspect the invention
relates to a
bioreactor construct comprising antibody secreting cells and a cell monolayer
in contact with
a surface of a plurality of hollow capillary tubes arranged in a parallel
array within a tubular
cartridge defining an intracapillary (IC) space and an extracapillary (EC)
space of the
bioreactor, each hollow capillary tube constructed of a semi-permeable
membrane defining
an internal surface adjacent to the IC space and an external surface adjacent
to the EC space,
wherein the cell monolayer is in contact with the internal or external surface
of the plurality
of capillary tubes.
In embodiments, the internal or external surface of the plurality of capillary
tubes is
coated with one or more extracellular matrix components and the cell monolayer
is in contact
with the coated surface. In embodiments, the one or more extracellular matrix
components is
selected from the group consisting of collagen IV, laminin, entactin,
tenascin, and
fibronectin. In embodiments, a surface of each hollow capillary tube is coated
with a mixture
of collagen IV and laminin I. In a further embodiment, the internal or
external surface of the
plurality of capillary tubes is coated with one or more of a natural polymer,
a biocompatible
synthetic polymer, a synthetic peptide, or a composite derived from any
combination thereof.
In embodiments, the natural polymer is selected from the group consisting of
agarose,
alginate, cellulose, chitosan, gelatin, and mixtures thereof. In embodiments,
the
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biocompatible synthetic polymer is selected from the group consisting of
acrylate polymers,
polyethylene co-vinyl acetate, polyethylene glycol, polysulfone, polyvinyl
alcohol,
polyvinylidene fluoride, sodium polyacrylate, mixtures thereof.
In embodiments, the semi-permeable membrane is fabricated from polyvinylidene
difluoride (PVDF) or polysulfone. In embodiments, the semi-permeable membrane
has a
molecular weight cut-off (MWCO) between 5-80 kilodaltons (kDa).
In embodiments, the cell monolayer comprises primary cells or immortalized
cells. In
embodiments, the cell monolayer comprises mammalian cells. In embodiments, the
mammalian cells are human, bovine, goat, sheep, canine, porcine, rodent, or
non-human
primate cells. In embodiments, the mammalian cells are human cells.
In embodiments, the cell monolayer comprises epithelial cells, fibroblast
cells, or a
combination thereof. In embodiments, the cell monolayer comprises mammary
epithelial
cells and optionally, fibroblast cells.
In embodiments, the cell monolayer comprises cells genetically engineered to
express
one or more proteins selected from the group consisting of CD40, polymeric
immunoglobulin
receptor (pIgR), and immunoglobulin J-chain.
In embodiments, the antibody secreting cells are primary B lymphocytes. In
embodiments, the primary B lymphocytes are obtained from a peripheral blood
mononuclear
cell (PBMC) fraction, mammoplasty tissue, or mammalian milk. In embodiments,
the
antibody secreting cells are immortalized cells selected from the group
consisting of
lymphoblasts, B lymphocytes, and hybridomas. In embodiments, the antibody
secreting cells
are genetically engineered to express BCL-6/BCL-XL.
In embodiments, the antibody secreting cells are human cells and the cell
culture
medium comprises one or more B cell activating molecules selected from the
group
consisting of anti-CD40 antibody, anti-IgM antibody, IL-4, IL-2, and IL-10.
In embodiments, the bioreactor further comprises T helper cells.
In another aspect, the invention relates to methods for culturing antibody
secreting
cells, the method comprising seeding a bioreactor construct with antibody
secreting cells and
a second type of cell selected from epithelial cells, fibroblast cells, or a
combination thereof;
wherein the bioreactor construct comprises a surface of a plurality of hollow
capillary tubes
arranged in a parallel array within a tubular cartridge defining an
intracapillary (IC) space
and an extracapillary (EC) space of the bioreactor, each hollow capillary tube
constructed of
a semi-permeable membrane defining an internal surface adjacent to the IC
space and an
external surface adjacent to the EC space; wherein the second cell type forms
a monolayer in
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contact with the internal or external surface of the plurality of capillary
tubes; and wherein a
cell culture medium fills the IC space or the EC space, or both.
In accordance with the methods described here, the bioreactor construct, cell
monolayer, and antibody secreting cells are as described herein.
In embodiments, the methods further comprise a step of preparing the
bioreactor prior
to seeding the cells, wherein preparing the bioreactor comprises coating an
internal or
external surface of the plurality of capillary tubes with one or more
extracellular matrix
components followed by seeding the cells into the bioreactor such that the
seeded cells are in
contact with the coated surface. In embodiments, the one or more extracellular
matrix
components is selected from the group consisting of collagen IV, laminin,
entactin, tenascin,
and fibronectin. In embodiments, the one or more extracellular matrix
components comprises
a mixture of collagen IV and laminin I.
In a further aspect, the invention relates to immunoglobulin molecules
produced by
antibody secreting cells cultured according to the methods described herein.
In embodiments,
the immunoglobulin molecule is a secretory IgA, secretory IgM, or IgG, or a
mixture of any
of the foregoing, for example a polyclonal mixture. In embodiments, the IgG
may be selected
from any subtype, for example the IgG subtype may be selected from the group
consisting of
IgA 1 , IgA2, IgGl, IgG2, IgG3, IgG4, and allotypes; or a polyclonal mixture
thereof.
In a further aspect, the invention relates to compositions comprising an
immunoglobulin molecule produced in the bioreactor construct in accordance
with the
methods described here. In embodiments, the composition comprises a secretory
IgA. In
embodiments, the composition comprises milk or a milk product. In embodiments,
the milk is
human, bovine, goat, or sheep milk.
These and other aspects of the invention are set forth in more detail in the
description
of the invention below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an electron micrograph of secretory IgA dimers isolated from the
supernatant of cells cultured as described in Example 1.
FIG. 2 shows increasing production of immunoglobulin molecules over time as
produced in culture with mammary epithelial cells as described in Example 1.
FIG. 3A-C shows tandem mass spectrophotography histograms of immunoglobulin
and immunoglobulin receptor peptides identified in the supernatant of cells
cultured as
described in Example 1. A, Immunoglobulin gamma-1 heavy chain (304-319); B,
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Immunoglobulin heavy constant alpha 1 (283-299); C, Polymeric immunoglobulin
receptor
(623-638).
FIG 4. Shows a representative chromatograph of a sample of the supernatant of
cells
cultured as described in Example 1. Light grey line represents the full
spectrum, which is
overlaid with the spectra of individual proteins isolated from the sample,
shown as black
lines. Arrows indicate immunoglobulin and immunoglobulin receptor peaks.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods for culturing antibody secreting
cells with a
second type of cell forming a monolayer in a hollow fiber bioreactor. In a
further aspect, the
invention relates to a bioreactor construct comprising antibody secreting
cells and a cell
monolayer on a plurality of hollow capillary tubes arranged in a parallel
array within a
tubular cartridge defining an intracapillary (IC) space and an extracapillary
(EC) space of the
bioreactor. In a further aspect, the invention relates to immunoglobulin
molecules produced
by antibody secreting cells cultured in accordance with the methods described
here, and
related compositions.
In accordance with the present invention, antibody secreting cells are co-
cultured with
at least one additional cell type which forms a cell monolayer. The cell
monolayer preferably
comprises mammalian cells, e.g., human, bovine, goat, sheep, canine, porcine,
rodent, or non-
human primate cells. In one embodiment the cells are human cells. In
embodiments, the cell
monolayer comprises fibroblast or epithelial cells, or a combination of
fibroblast and
epithelial cells.
The cells of the monolayer may be primary cells or immortalized cells.
In embodiments, the cell monolayer comprises cells genetically engineered to
express one or
more proteins to enhance antibody production by the antibody secreting cells.
In
embodiments, the one or more proteins is selected from the group consisting of
cluster of
differentiation 40 (CD40), polymeric immunoglobulin receptor (pIgR), and
immunoglobulin
J-chain. Methods for genetic engineering known in the art can be used to
introduce the genes
encoding these proteins.
Suitable methods for genetic engineering include technologies utilizing
nucleases for
genome editing, such as zinc finger nucleases, transcription activator-like
effector nuclease,
and CRISPR/Cas9. See for example Koch et al., Nat Protoc. 2018 Jun; 13(6):
1465-1487;
and a review of CRISPR/Cas systems in Nat Biotechnol 2014 Apr;32(4):347-55.
More
traditional recombinant technologies which utilize bacterial or viral vectors
may also be used
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and are described, for example, in Green and Sambrook, Molecular Cloning Cold
Spring
Harbor Laboratory Press 2012. See e.g., chapters 3-5, and 15, 16.
In embodiments, the cell monolayer comprises mammary epithelial cells. In
embodiments, the mammary epithelial cells are milk producing cells. In
embodiments, the
milk producing mammary epithelial cells are primary cells, or primary
immortalized cells.
The antibody secreting cells may also be primary or immortalized cells. In
embodiments, the antibody secreting cells are primary B lymphocytes. Primary B
lymphocytes may be obtained according to methods known in the art, for example
from a
peripheral blood mononuclear cell (PBMC) fraction, mammoplasty tissue, or
mammalian
milk.
In embodiments, the antibody secreting cells are immortalized cells selected
from the
group consisting of lymphoblasts, B lymphocytes, and hybridomas. In
embodiments, the
methods described here encompass immortalizing a primary cell.
Methods for immortalizing cells are known in the art. For example, cells may
be
immortalized by introduction of a virus or viral gene, e.g., using Epstein
Barr virus, 5V40
virus, human papillomavirus (HPV) sequences, human T-lymphotropic virus type 1
(HTLV-
1); by genetically engineering the cells to express certain proteins, such as
BCL-6/BCL-XL,
TERT, or telomerase; or by fusing the cells with an immortal cell line to form
a hybridoma.
Combinations of the foregoing techniques may also be used. In embodiments, the
cells are
immortalized through genetic engineering to express BCL-6/BCL-XL.
In embodiments, the antibody secreting cells are genetically engineered to
express
one or more molecules selected from the group consisting of a F(ab) fragment,
a polymeric
immunoglobulin receptor (pIgR), and an immunoglobulin J-chain. In embodiments,
the
antibody secreting cells are genetically engineered to delete the gene
encoding activation-
induced cytidine deaminase (AID), the AICDA gene.
In embodiments, the antibody secreting cells are primary B lymphocytes,
preferably
isolated from milk, blood, or mammoplasty tissue, preferably human but can be
from other
mammalian species e.g., bovine, goat, sheep, canine, porcine, rodent, or non-
human primate.
In embodiments the primary B lymphocytes are genetically engineered to express
one or
more molecules including a F(ab) fragment with desired affinity, a polymeric
immunoglobulin receptor (pIgR), and an immunoglobulin J-chain; or to delete
one or more
genes, such as the AICDA gene. In a further embodiment, the genetically
engineered primary
B lymphocytes are immortalized. In embodiments, the primary cells are
immortalized using
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hybridoma technology or by further genetically engineering the cells to
express a suitable
protein, such as BCL-6/BCL-XL, TERT, or telomerase.
Methods for genetic engineering of primary B lymphocytes are described, for
example, in Johnson et al., Sci Rep 8, 12144 (2018).
As used in the description of the invention and the appended claims, the
singular
forms "a," "an" and "the" are intended to include the plural forms as well,
unless the context
clearly indicates otherwise.
As used herein, "and/or" refers to and encompasses any and all possible
combinations
of one or more of the associated listed items, as well as the lack of
combinations when
interpreted in the alternative ("or").
As used herein, the term "about," as used herein when referring to a
measurable value
such as an amount of a compound or agent of this invention, dose, time,
temperature, and the
like, is meant to encompass variations of less than 1-5% of the specified
amount.
As used herein, the transitional phrase "consisting essentially of' is to be
interpreted
as encompassing the recited materials or steps and those that do not
materially affect the
basic and novel characteristic(s) of the claimed invention. This term is not
equivalent to the
open-ended "comprising."
As used herein, the term "protein" encompasses peptides, polypeptides and
proteins,
unless indicated otherwise.
As used herein, by "isolate" (or grammatical equivalents, e.g., "extract") a
product, it
is meant that the product is at least partially separated from at least some
of the other
components in the starting material.
As used herein, the term "genetically modified or engineered" encompasses
materials
produced by recombinant technology.
Bioreactor Constructs
The present invention relates to bioreactor constructs comprising antibody
secreting
cells and a second type of cell forming a monolayer, as discussed above. The
bioreactor
construct consists of at least one tubular cartridge housing a plurality of
hollow capillary
tubes arranged in a parallel array within the cartridge and defining an
intracapillary (IC)
space and an extracapillary (EC) space of the bioreactor construct. Each
hollow capillary tube
is constructed of a semi-permeable membrane defining an internal surface
adjacent to the IC
space and an external surface adjacent to the EC space. In embodiments, the
capillaries are
fabricated from PVDF, polysulfone, or other biologically suitable materials.
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In embodiments, before seeding cells into the bioreactor, the bioreactor
capillaries are
first coated with an extracellular matrix (ECM) material. The ECM may comprise
one or
more ECM components selected from the group consisting of chondroitin sulfate,
collagen
IV, elastin, fibronectin, heparan sulfate, hyaluronic acid, keratin sulfate,
laminin, nidogen-1
(NlD-1, also known as entactin) and tenascin. In embodiments, the ECM
comprises or
consists of a 1:1 mixture of collagen IV and laminin I in PBS.
In embodiments, before seeding cells into the bioreactor, the bioreactor
capillaries are
first coated with one or more of a natural polymer, a biocompatible synthetic
polymer, a
synthetic peptide, or a composite derived from any combination thereof.
Suitable natural
polymers include agarose, alginate, cellulose, chitosan, and gelatin. Suitable
biocompatible
synthetic polymers include acrylate polymers, polyethylene co-vinyl acetate,
polyethylene
glycol, polysulfone, polyvinyl alcohol, polyvinylidene fluoride, sodium
polyacrylate, and
mixtures thereof.
Methods
The present invention provides methods for co-culturing antibody secreting
cells with
a second type of cell that forms a monolayer on a surface of the capillary
tubes within a
hollow fiber bioreactor. Thus, the methods employ a hollow fiber bioreactor
construct, as
described above.
Standard conditions suitable for the culture of mammalian cells may be used in
co-
culturing the antibody secreting cells with a second type of cell, as
described herein. For
example, a controlled environment within the bioreactor construct suitable for
culture of
mammalian cells may comprise a temperature in the range of 35-39 C,
preferably about 37
C; in an atmosphere of 4-6 % CO2, preferably about 5 % CO2. The cells are
cultured in a
minimum or basal" cell growth medium which includes a carbon source, a
buffering system
to maintain a neutral pH, one or more essential amino acids, one or more
vitamins and/or
cofactors, and one or more inorganic salts.
Any suitable buffering system may be used for the basal growth medium. An
exemplary system is sodium bicarbonate and/or 4-(2-hydroxyethy1)1-
piperazineethanesuifonic acid (HEPES).
In embodiments, the basal culture medium comprises one or more essential amino
acids in an amount from about 0.5-5 mM. In some embodiments, the one or more
essential
amino acids is selected from arginine and cysteine, or both. In embodiments,
the basal culture
medium comprises one or more vitamins and/or cofactors in an amount from about
0.01-50
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M. In some embodiments, one or more vitamins and/or cofactors is selected from
thiamine
and riboflavin, or both. In embodiments, the basal culture medium comprises
one or more
inorganic salts in an amount from about 100-150 mg/L. In some embodiments, the
one or
more inorganic salts is selected from calcium and magnesium, or both.
Suitable basal cell growth mediums are known in the art and include, for
example,
Ames Medium, Basal Media Eagle, Minimum Essential Medium Eagle (MEM),
Dulbecco's
Modified Eagle's Media (DMEM), Iscove's Modified Dulbecco's Medium (IMDM). In
some
embodiments, the basal cell growth medium is DMEM supplemented with a
chemically
defined medium for high density cell culture, such as FiberCell Systems CDM-
HD.
In embodiments, the basal cell growth medium is supplemented with one or more
growth factors, hormones, or B cell activating molecules. In embodiments, the
basal cell
growth medium is supplemented with one or more of epidermal growth factor,
prolactin, and
a B cell activating molecule selected from the group consisting of anti-CD40
antibody, anti-
IgM antibody, IL-4, IL-2, and IL-10, and combinations thereof.
In embodiments, both the antibody secreting cells and the second cell type are
seeded
together into the bioreactor. In embodiments, the antibody secreting cells are
seeded after the
second cell type. In embodiments, the methods described here may further
comprise seeding
additional antibody secreting cells into the bioreactor at one or more times
following the
initial seeding.
In embodiments, cells are seeded into the extracapillary (EC) space, and a
basal
medium is pumped through the capillaries, into the intracapillary space (IC).
In an alternative
embodiment, the cells are seeded into the IC space and the EC space contains
the basal
medium.
Prior to seeding the cells in the bioreactor, it is generally advantageous to
culture the
cells in order to obtain an expanded population of cells in a phase of
exponential growth.
Accordingly, the methods described here may further comprise a step of
culturing the
antibody secreting cells and/or the second cell type prior to seeding the
cells into the
bioreactor in order to obtain an expanded population of cells, preferably in a
phase of
exponential growth at the time of seeding. Cells are seeded into the
bioreactor using standard
cell culture protocols.
The second cell type may be any type of cell capable of forming a monolayer on
a
surface of the capillaries, for example fibroblast or epithelial cells. The
monolayer forming
cells may be derived from any organ, including for example, mammary, lung,
skin, kidney,
colon, etc.
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In embodiments, the second cell type comprises mammary epithelial cells and
optionally, fibroblast cells. In embodiments, the methods further comprise
stimulating milk
production in the mammary epithelial cells, which may be primary cells or
immortalized cells,
including immortalized primary cells. In an embodiment, stimulating milk
production
comprises contacting a confluent monolayer of the mammary epithelial cells
with a first
amount of prolactin and culturing the cells for a period of time; followed by
contacting the
cells with a second amount of prolactin and culturing the cells for a second
period of time. In
embodiments, the first amount of prolactin is from about 80-150 ng/ml,
preferably about 100
ng/ml; and the second amount of prolactin is from about 150-250 ng/ml,
preferably about 200
ng/ml, supplemented in the basal culture medium. In embodiments, the first
period of time is
from about 3-15 days; and the second period of time is from about 5-20 days.
The formation of a cell monolayer in the bioreactor can be monitored, for
example, by
determining glucose utilization over a period of days following seeding the
cells into the
bioreactor. Glucose utilization is an indicator of cellular metabolism. During
exponential
growth, glucose utilization increases sharply, then slows and drops to a lower
steady state
when the cells reach confluence. Confluent cells will form a barrier dividing
the
intracapillary (IC) and extracapillary (ECS) spaces. The integrity of the
monolayer may be
determined, for example, using a transepithelial electrical resistance (TEER)
assay. TEER
measures a voltage difference between the fluids in the two compartments.
Where barrier
integrity is lost, and two fluids mix, the voltage difference drops to zero.
Conversely, a
voltage difference indicates that the barrier is intact.
In accordance with the methods described here, in some embodiments the basal
cell
culture medium of the bioreactor is supplemented with one or more B cell
activating
molecules selected from the group consisting of anti-CD40 antibody, anti-IgM
antibody, IL-
4, IL-2, and IL-10. These and similar agents are commercially available, for
example from
Sigma Aldrich, Irvine Scientific, and Schering Plough. Suitable methods for
the in vitro
activation of antibody secreting B cells are described, for example, in
Lefevre et al. J
Immunol 1999; 163:1119-1122.
In an embodiment, the methods described here can be used to produce fully
human
antibodies, either with or without genetic modifications. In addition, the
methods described
here provide improvements in antibody production attainable due to the ability
to continuous
monitor the amount and types of antibodies present by sampling of the
bioreactor supernatant
through the port. Through such continuous monitoring, it is possible to
control both the
overall production of antibodies as well as the ratios of monoclonal and
polyclonal mixtures.
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in
For example, additional antibody secreting cells may be added to maintain or
achieve a target
formulation containing a specific ratio of neutralizing antibodies for
therapeutic use.
In embodiments, the antibody secreting cells are human cells secreting
neutralizing
antibodies against an antigenic molecule of a pathogen, such as a virus or
bacteria. In
embodiments, the antigenic molecule is the spike protein of SARS-CoV-2. In
embodiments,
the neutralizing antibodies are monoclonal antibodies. In embodiments, the
neutralizing
antibodies are polyclonal antibodies. Methods for producing human cells
secreting
neutralizing antibodies against a target antigenic molecule (also referred to
as an
'immunogen') are known in the art and can be used to produce suitable antibody
secreting
cells for use in the methods described here. For example, as described in
Boonyaratanakornit
and Taylor, Front. Immunol 2019.
In embodiments, the present methods further provide a fully human system for
the
production of antibodies that is free of endotoxin and other impurities, such
that the
antibodies produced according to the methods described here do not require the
post-
processing steps typically needed to remove such contaminants prior to use in
humans. This
is achieved in part by ensuring that all components of the bioreactor system
are "food grade"
in accordance with the definition of regulatory authorities, such as the US
Food and Drug
Administration. Thus, in embodiments, the basal culture medium, ECM materials,
etc. of the
bioreactor system are comprised of food grade ingredients.
In embodiments, the cell monolayer is a milk producing mammary epithelial cell
monolayer and the components of the bioreactor system are food grade
materials, including
without limitation the basal culture medium, media supplements, and the ECM
materials. In
accordance with this embodiment, the invention further provides a food grade
biosynthetic
milk product containing antibodies. In embodiments, the antibody containing
biosynthetic
milk product is unpasteurized. In embodiments, the invention further provides
a composition
comprising the antibody containing biosynthetic milk product.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. The terminology used in the description of the invention herein is
for the purpose of
describing particular embodiments only and is not intended to be limiting of
the invention.
Except as otherwise indicated, standard methods known to those skilled in the
art may
be used for production of recombinant molecules, manipulation of nucleic acid
sequences,
production of transformed cells, the construction of viral vector constructs,
and transiently
and stably transfected packaging cells. Such techniques are known to those
skilled in the art.
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See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed.
(Cold Spring
Harbor, NY, 1989); F. M. Ausubel et al. Current Protocols In Molecular Biology
(Green
Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).
All publications, patent applications, patents, nucleotide sequences, amino
acid
sequences and other references mentioned herein are incorporated by reference
in their
entireties for the teachings relevant to the sentence and/or paragraph in
which the reference is
presented.
Having described the present invention, the same will be explained in greater
detail in
the following examples, which are included herein for illustration purposes
only, and which
are not intended to be limiting to the invention.
EXAMPLES
EXAMPLE 1
The following describes the cultivation of primary antibody secreting B cells
in a
bioreactor seeded with primary human mammary epithelial cells (HUMECs). In
this
example, the antibody secreting B cells were not separately seeded into the
bioreactor. As
discussed below, biochemical analyses of the supernatant of the bioreactor
identified
numerous immunoglobulin molecules, including secretory IgA dimers,
demonstrating the
presence of antibody secreting B cells.
Preparation of hollow fiber bioreactor:
Prior to seeding with cells, the bioreactor cartridge was prepared by
incubation with
PBS for a minimum of 24 hours followed by coating the capillaries with a 1:1
mixture of
collagen IV and laminin I (25 1.ig Laminin-111, 25 1.ig Collagen IV) in PBS at
room
temperature overnight. The collagen/laminin mixture was then exchanged with
cell growth
medium and incubated overnight at room temperature.
Culture of cells in the bioreactor:
Primary human mammary epithelial cells (HUMECs) were obtained from the
American Type Culture Collection (ATCCO) and expanded in accordance with
standard
protocls before seeding into the bioreactor. After seeding, cells were allowed
to proliferate
until confluence was reached, as determined by glucose utilization. Cells were
cultured in a
basal growth medium (ATCC PCS600030TM) until confluence. Following
confluence, the
basal medium was supplemented with Dulbecco's Modified Eagle's Medium (DMEM,
Sigma Aldrich) containing a chemically defined medium for high density cell
culture
(FiberCellSystems CDM-HD, 10% by vol. in basal medium).
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Milk production was stimulated with 100 ng/ml prolactin followed by increasing
prolactin to 200 ng/ml.
Samples of culture supernatant were collected from the bioreactor port hole
for
subsequent analyses.
Characterization of antibody-secreting B cells
The presence of antibody secreting B cells in the bioreactor is evidenced by
the
detection of various immunoglobulin molecules in samples collected from the
bioreactor.
Immunoglobulin molecules were detected by liquid chromatography coupled with
mass
spectrophotometry (LC-MS) and by tandem mass spectrophotometry (MS-MS) using
standard protocols. Representative MS chromatographs of proteins isolated from
a sample of
the supernatant of cells cultured as described here are shown in FIG 3A-C. The
figure
depicts peptides representing immunoglobulin gamma-1 heavy chain (304-319),
immunoglobulin heavy constant alpha 1 (283-299), and polymeric immunoglobulin
receptor
(623-638), respectively. A representative chromatograph showing peaks for the
whole sample
(light grey line) overlaid with the peaks from the three isolated proteins
(black filled areas) is
shown in FIG 4. Table 1 below lists all of the immunoglobulin molecules
identified in the
culture supernatant.
Table 1: Immunoglobulin molecules identified in bioreactor supernatant
Immunoglobulin alpha-2
PODOX2 I IGA2_HUMAN heavy chain
Immunoglobulin gamma-1
PODOX5 I IGG1_HUMAN heavy chain
Immunoglobulin heavy
P018771IGHA2_HUMAN constant alpha 2
Immunoglobulin heavy
P018571IGHG1_HUMAN constant gamma 1
Immunoglobulin heavy
P01859 I IGHG2_HUMAN constant gamma 2
Immunoglobulin heavy
P018601IGHG3_HUMAN constant gamma 3
Immunoglobulin heavy
P01861 I IGHG4_HUMAN constant gamma 4
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Immunoglobulin heavy
P018711IGHM_HUMAN constant mu
P0159111GLHUMAN Immunoglobulin J chain
Immunoglobulin lambda
PODOY2IIGLC2_HUMAN constant 2
Immunoglobulin lambda
PODOY3IIGLC3_HUMAN constant 3
Immunoglobulin lambda-like
B9A064 I I GLL5_H U MAN polypeptide 5
Immunoglobulin mu heavy
PODOX6IIGM_HUMAN chain
In addition, secretory IgA dimers were also identified as evidenced by the
electron
micrograph shown in FIG. 1.
In addition, production of secretory IgA dimers was quantified by use of a
colorimetric Human Secretory IgA ELISA Kit produced by Novus Biologicals.
ELISA
analysis demonstrated a peak production of 250 mg of SIgA per day per liter of
ECS, an
unprecedented level of efficiency for biosynthesis of secretory IgA dimers.
The production
of 250 mg/day/L-ECS was extrapolated from producing 8mg of sIgA over 10 days
in 3mL
ECS. The invention described herein surprisingly showed the high total amount
of SIgA
produced, e.g., 8 mg over 10 days, in a small volume.
Antibody secreting plasma B cells are notoriously short lived ex vivo when
isolated
from blood, milk or tissue, with reported half lives of 3-5 days without
molecular or genetic
modification. As shown in FIG. 2, the methods described here supported a much
longer-term
survival of non-modified antibody secreting cells than expected. In a typical
ex vivo plasma
cell culture, the antibody secreting cell population would be expected to be
depleted by about
day 10 of culture. Instead, in the system described here, around ex vivo day
51-58, antibody
production substantially increased. Without being bound by any particular
theory, co-culture
with epithelial monolayers as described here maximizes the survival of
antibody secreting
cells, as well as their production of antibody. These methods can be used to
produce, for
example, fully human polyclonal antibodies of any desired class or subtype at
therapeutic
scale, without the need to genetically modify the antibody secreting cells. Of
course, targeted
genetic modifications could also be used to further enhance the cells'
survival, their antibody
production, and antigen affinity, as described herein.
CA 03207250 2023-07-05
WO 2022/150482 PCT/US2022/011446
14
Production of antibodies at commercially feasible scale
The methods described here provide a proof-of-concept for the production of
antibodies using bioreactor cultured antibody secreting cells. The hollow
fiber bioreactor is a
particularly advantageous system for maximizing surface area while allowing
the cells to
organize into three dimensional structures. The process is readily scalable
for commercial
production. In the present example, a relatively small bioreactor cartridge
was used to culture
the epithelial monolayer. It contained about 400 square centimeter (cm2) of
surface area for
cell growth. Using a larger commercially available bioreactor cartridge would
substantially
increase the surface area and consequently the production of antibodies. For
example, larger
commercially available bioreactors may have a surface area of about 3 square
meters (m2).
The process is further scalable, for example, by packing more fibers and/or
longer fibers into
one or more cartridges aligned in parallel.
The foregoing examples are illustrative of the present invention, and are not
to be
construed as limiting thereof. Although the invention has been described in
detail with
reference to preferred embodiments, variations and modifications exist within
the scope and
spirit of the invention as described and defined in the following claims.
